A postsynaptic role for Rhp55/57 that is responsible for cell death in Deltarqh1 mutants following replication arrest in Schizosaccharomyces pombe

Abstract

Following replication arrest, multiple cellular responses are triggered to maintain genomic integrity. In fission yeast, the RecQ helicase, Rqh1, plays a critical role in this process. This is demonstrated in Deltarqh1 cells that, following treatment with hydroxyurea (HU), undergo an aberrant mitosis leading to cell death. Previous data suggest that Rqh1 functions with homologous recombination (HR) in recovery from replication arrest. We have found that loss of the HR genes rhp55(+) or rhp57(+), but not rhp51(+) or rhp54(+), suppresses the HU sensitivity of Deltarqh1 cells. Much of this suppression requires Rhp51 and Rhp54. In addition, this suppression is partially dependent on swi5(+). In budding yeast, overexpressing Rad51 (the Rhp51 homolog) minimized the need for Rad55/57 (Rhp55/57) in nucleoprotein filament formation. We overexpressed Rhp51 in Schizosaccharomyces pombe and found that it greatly reduced the requirement for Rhp55/57 in recovery from DNA damage. However, overexpressing Rhp51 did not change the Deltarhp55 suppression of the HU sensitivity of Deltarqh1, supporting an Rhp55/57 function during HR independent of nucleoprotein filament formation. These results are consistent with Rqh1 playing a role late in HR following replication arrest and provide evidence for a postsynaptic function for Rhp55/57.

Figure 1.—

HU and UV sensitivity of HR mutants alone and combined with Δrqh1. Double mutants between Δrqh1 and mutants of the RAD52 epistasis group were created. To measure HU sensitivity, cells were grown to midlog and then each single and double mutant was plated onto plates containing HU of varying concentrations and colonies were counted after 4–6 days of incubation at 30°. To measure UV sensitivity, midlog cultures were grown and cells were spread onto YEA plates at varying concentrations and irradiated with the indicated dose of 254 nm UV light. The results are shown. •, wild type; ▪, Δrqh1. (a) ▵, Δrhp51; ⋄, Δrqh1 Δrhp51. (b) ○, Δrhp54; ♦, Δrqh1 Δrhp54. (c) ▵, Δrhp55; ♦, Δrqh1 Δrhp55. (d) ▵, Δrhp51; ⋄, Δrqh1 Δrhp51; ○, Δrhp54; ▴, Δrqh1 Δrhp54. (e) ▵, Δrhp55; ♦, Δrqh1 Δrhp55. (Note that some error bars are smaller than the symbols.)

Figure 1.—

HU and UV sensitivity of HR mutants alone and combined with Δrqh1. Double mutants between Δrqh1 and mutants of the RAD52 epistasis group were created. To measure HU sensitivity, cells were grown to midlog and then each single and double mutant was plated onto plates containing HU of varying concentrations and colonies were counted after 4–6 days of incubation at 30°. To measure UV sensitivity, midlog cultures were grown and cells were spread onto YEA plates at varying concentrations and irradiated with the indicated dose of 254 nm UV light. The results are shown. •, wild type; ▪, Δrqh1. (a) ▵, Δrhp51; ⋄, Δrqh1 Δrhp51. (b) ○, Δrhp54; ♦, Δrqh1 Δrhp54. (c) ▵, Δrhp55; ♦, Δrqh1 Δrhp55. (d) ▵, Δrhp51; ⋄, Δrqh1 Δrhp51; ○, Δrhp54; ▴, Δrqh1 Δrhp54. (e) ▵, Δrhp55; ♦, Δrqh1 Δrhp55. (Note that some error bars are smaller than the symbols.)

Figure 2.—

Evidence that HR intermediates accumulate in HU-treated Δrqh1 cells that are suppressed by Δrhp55. Previous studies had shown that aberrant mitosis occurs in Δrqh1 cells following replication arrest. We speculated that torn and unevenly distributed nuclear material was due to unresolved recombinant intermediates. We tested this hypothesis by determining if loss of rhp55⁺ could suppress this phenotype. (a) Examples of DAPI-stained cells visualized by fluorescent microscopy are shown following HU treatment and a 3-hr recovery. Arrowheads point to septa of dividing cells. Asterisks indicate cut chromosomes. The double asterisk indicate cells where all DNA segregated into one daughter cell. (b) Quantitation of the number of cells with aberrant chromosomes visible following DAPI staining after a 3-hr release from replication block. (c) PFGE was used as another way of monitoring the fate of chromosomes following HU treatment. Replication fork structures and recombination intermediates are inhibited from exiting the well. We compare the chromosomes from Δrqh1 cells with those from wild type, Δrhp55, and Δrqh1 Δrhp55. Lanes 1, 6, 11, and 16, chromosomes from cycling cells; lanes 2, 7, 12, and 17, chromosomes from cells exposed to 15 mm HU for 5 hr; lanes 3, 8, 13, and 18, chromosomes from cells 2 hr after release; lanes 4, 9, 14, and 19, chromosomes from cells 4 hr after release; lanes 5, 10, 15, and 20, chromosomes from cells 6 hr after release.

Figure 2.—

Evidence that HR intermediates accumulate in HU-treated Δrqh1 cells that are suppressed by Δrhp55. Previous studies had shown that aberrant mitosis occurs in Δrqh1 cells following replication arrest. We speculated that torn and unevenly distributed nuclear material was due to unresolved recombinant intermediates. We tested this hypothesis by determining if loss of rhp55⁺ could suppress this phenotype. (a) Examples of DAPI-stained cells visualized by fluorescent microscopy are shown following HU treatment and a 3-hr recovery. Arrowheads point to septa of dividing cells. Asterisks indicate cut chromosomes. The double asterisk indicate cells where all DNA segregated into one daughter cell. (b) Quantitation of the number of cells with aberrant chromosomes visible following DAPI staining after a 3-hr release from replication block. (c) PFGE was used as another way of monitoring the fate of chromosomes following HU treatment. Replication fork structures and recombination intermediates are inhibited from exiting the well. We compare the chromosomes from Δrqh1 cells with those from wild type, Δrhp55, and Δrqh1 Δrhp55. Lanes 1, 6, 11, and 16, chromosomes from cycling cells; lanes 2, 7, 12, and 17, chromosomes from cells exposed to 15 mm HU for 5 hr; lanes 3, 8, 13, and 18, chromosomes from cells 2 hr after release; lanes 4, 9, 14, and 19, chromosomes from cells 4 hr after release; lanes 5, 10, 15, and 20, chromosomes from cells 6 hr after release.

Figure 3.—

Suppression of Δrqh1 HU sensitivity depends on the function of Δrhp51 and Δrhp54. (a and b) Tenfold serial dilutions of each strain were spotted onto plates containing either 0 mm or 3.6 mm HU. Plates were incubated for 5 days at 30° and photographed. (c) At reduced temperatures Δrhp55 mutants show reduced resistance to HU but maintain their suppression of Δrhq1. The plates contain 2.4 mm HU and were incubated at 30° for 5 days or 22° for 8 days.

Figure 3.—

Suppression of Δrqh1 HU sensitivity depends on the function of Δrhp51 and Δrhp54. (a and b) Tenfold serial dilutions of each strain were spotted onto plates containing either 0 mm or 3.6 mm HU. Plates were incubated for 5 days at 30° and photographed. (c) At reduced temperatures Δrhp55 mutants show reduced resistance to HU but maintain their suppression of Δrhq1. The plates contain 2.4 mm HU and were incubated at 30° for 5 days or 22° for 8 days.

Figure 4.—

Overexpression of Rhp51. Full-length rhp51⁺ was cloned into the pREP81x vector (pREP 81x-Rhp51), transformed into various strains, and tested for HU sensitivity. (a) Wild-type and Δrhp55 cells containing pREP 81x-Rhp51 were grown to midlog in media either containing 8 mm thiamine or lacking thiamine. These cells were then irradiated with varying doses of γ-rays and subsequently plated onto YEA plates containing 8 mm thiamine. (b) Wild-type (WT), Δrhp55, Δrqh1, and Δrhp55 Δrqh1 strains were transformed with pREP 81x-Rhp51. Cells were grown to midlog in media either containing 8 mm thiamine or lacking thiamine. Then 15 mm HU was added to each culture and allowed to incubate for an additional 9 hr. Samples were then collected and plated onto YEA plates with 8 mm thiamine. The plates were incubated for 4 days and colonies were counted. (c) Extracts were prepared from wild-type and Δrhp55 Δrqh1 cells all containing pREP 81x-Rhp51 with cultures grown in the presence or absence of thiamine. A total of 150 μg of each extract was loaded onto a 12% PAGE SDS gel and the samples were separated by electrophoresis. As a control, 100 μg of nuclear extract prepared from HeLa cells was also loaded onto the gel. The gel was blotted and Rhp51 was detected using an antibody against human Rad51, which cross-reacts with S. pombe Rhp51. Antibody binding was detected by chemiluminescence.

Figure 5.—

Suppression of Δrqh1 HU sensitivity by Δrhp55 is partially dependent on Swi5. We investigated the possibility that Swi5 was necessary for the suppression of the HU sensitivity of Δrqh1 by Δrhp55. (a) Serial dilutions of midlog cultures of wild-type, Δrhp55, Δswi5, Δrhp51, and Δrhp55 Δswi5 cells were plated onto YEA or YEA containing 2.4 mm HU followed by incubation for 5 days. (b) Serial dilutions of midlog cultures of wild-type, Δrqh1, Δswi5, and Δrqh1 Δswi5 cells were plated onto YEA or YEA containing 2.4 mm HU followed by incubation for 5 days. (c) Midlog cultures of wild type, Δrhp55, Δswi5, Δrqh1, Δrqh1 Δrhp55, and Δrqh1 Δrhp55 Δswi5 were plated onto YEA plates containing varying concentrations of HU and incubated for 4–6 days before colonies were counted.

Figure 6.—

The IR sensitivities of double mutants between Δrqh1 and HR genes show survival patterns similar to their HU sensitivities. The IR sensitivities of Δrhp55, Δrhp51, Δrqh1, Δrqh1 Δrhp55, and Δrqh1 Δrhp51 were tested. Cells from midlog cultures were plated onto YEA plates and irradiated with the indicated dose. Colonies were counted after 5 days. •, wild type; ▪, Δrqh1. (a) ▴, Δrhp55; ▵, Δrqh1 Δrhp55. (b) ▴, Δrhp51; ▵, Δrqh1 Δrhp51; ⋄, Δrqh1 Δrhp55 Δrhp51.